Transgenic plants synthesizing high amylose starch

ABSTRACT

Transgenic plant cells and plants are described which synthesize a starch which is modified in comparison with corresponding wild type plant cells and plants. The plant cells and plants described show a reduced activity of R1, BEI and BEII proteins. Furthermore, modified starches as well as methods for their production are described.

FIELD OF THE INVENTION

The present invention relates to genetically modified plant cells andplants wherein the genetic modification leads to the reduction of theactivity of R1 and BEI and BEII proteins in comparison withcorresponding plant cells of wild type plants that have not beengenetically modified. Furthermore, the present invention relates tomeans and methods for the production thereof. Plant cells and plants ofthat type synthesise a modified starch characterised in that it has anamylose content of at least 75% and—in comparison with starch ofcorresponding wild type plants which have not been geneticallymodified—a reduced phosphate content and/or a modified distribution ofthe side chains and/or an increased gel strength in the texture analyserand/or a modified starch granule morphology and/or a modified averagestarch granule size. Thus, the present invention also relates to starchthat can be synthesised by the plant cells and plants of the inventionas well as methods for the production of this starch.

With regard to the increasing importance of plant ingredients asrenewable raw material sources in the past few years, one of theproblems of research in the field of biotechnology is to endeavouradjustment of these raw materials to the requirements of the processingindustry. For allowing an application of renewable raw materials in asmany as fields as possible, it is furthermore necessary to achieve agreat variety of substances.

Apart from oils, fats and proteins, polysaccharides represent theessential renewable raw materials from plants. Among thepolysaccharides, starch plays a central role beside cellulose. It is oneof the most important storage substances in higher plants. For allowingas wide an application of starch as possible, it seems desirable toprovide plants which are able to synthesise modified starch that isparticularly suitable for different purposes. One possibility ofproviding such plants is—apart from cultivating—the purposeful geneticmodification of the starch metabolism of starch-producing plants bygenetic engineering.

The polysaccharide starch is a polymer of chemically uniform basicbuilding blocks—the glucose molecules. It is, however, a very complexmixture of different molecule forms which differ with regard to theirpolymerisation degree and the occurrence of branchings of the glucosechains. Thus, starch is not a uniform raw material. There are twochemically different components of starch: the amylose and theamylopectin. In plants typically used for the production of starch, suchas e.g. maize, wheat or potato, the synthesised starch consists of about20%–30% of amylose starch and of about 70%–80% of amylopectin starch.

Amylose was considered a linear polymer for a long time, consisting ofα-1,4-glycosidically bound α-D-glucose monomers. In recent studies,however, the presence of about 0.1% α-1,6-glycosidic branching pointshas been proven (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1–10;Takeda et al., Carbohydr. Res. 132, (1984), 83–92).

As a rule, the complete separation of the amylose from the amylopectinis very difficult so that the quality of the amylose strongly depends onthe type of the separation method chosen.

There are different methods for the determination of the amylosecontent. Some of these methods are based on the iodine-binding capacityof the amylose which can be determined potentiometrically (Banks &Greenwood, in W. Banks & C. T. Greenwood, Starch and its components(page 51–66), Edinburgh, Edinburgh University Press), amperometrically(Larson et al., Analytical Chemistry 25(5), (1953), 802–804) orspectrophotometrically (Morrison & Laignelet, J. Cereal Sc. 1, (1983),9–20). The determination of the amylose content can also be carried outcalorimetrically by means of DSC (differential scanning calorimetry)measurements (Kugimiya & Donovan, Journal of Food Science 46, (1981),765–770; Sievert & Holm, Starch/Stärke 45 (4), (1993), 136–139).Moreover, it is possible to determine the amylose content by using theSEC (size exclusion chromatography) of native or debranched starch. Thismethod was particularly recommended for the determination of the amylosecontent of genetically modified starches (Gérard et al., CarbohydratePolymers 44, (2001), 19–27).

The choice of the analysis method used for the determination of theamylose content of a starch has a crucial influence on the size of theamylose figures determined as could be shown by various studies (Shi etal., J. Cereal Science 27, (1998), 289–299; Gérard et al., CarbohydratePolymers 44, (2001), 19–27).

In contrast to the amylose, the amylopectin is branched to a largerdegree and exhibits about 4% branching points which occur due to thepresence of additional α-1,6-glycosidic linkings. The amylopectin is acomplex mixture of glucose chains branched differently.

A further essential difference between amylose and amylopectin is themolecular weight. While amylose—depending on the origin of thestarch—has a molecular weight of 5×10⁵−10⁶ Da, the molecular weight ofamylopectin is between 10⁷ and 10⁸ Da. Both macromolecules can bedifferentiated from each other by their molecular weight and theirdifferent physico-chemical properties, which can be made apparent in thesimplest way by their different iodine-binding properties.

The functional properties of the starch are strongly influenced—apartfrom the amylose/amylopectin ratio and the phosphate content—by themolecular weight, the pattern of the side chain distribution, thecontent of ions, the lipid and protein content, the average starchgranule size and the starch granule morphology etc. Important functionalproperties to be mentioned are, for example, the solubility, theretrogradation behaviour, the water binding capacity, the film formationproperties, the viscosity, the pasting properties, thefreeze-thaw-stability, the acid stability, the gel strength etc. Thestarch granule size, too, can be important for different applications.

The ratio of amylopectin and amylose has a strong influence on thephysico-chemical properties of the starches and, thus, on the possibleapplications of these starches. Since methods for the separation ofthese two components are very time-consuming and costly, such methodsare no longer used on a large technical scale (Yound, A. H. in: StarchChemistry and Technology. Eds. R. L. Whistler, J. N. BeMiller and E. F.Paschall. Academic Press, New York, 1984, 249–283). For a plurality ofapplications it would be desirable to have starches at disposal whichstill contain only one of the two polymers or at least one of the twostarch components in an enriched form.

So far, both mutants and plants produced by genetic engineering havebeen described which, in comparison with corresponding wild type plants,exhibit a modified amylopectin/amylose ratio.

For example, the so-called “waxy” mutant from maize exhibiting amutation in the gene encoding the starch granule bound starch synthase I(abbreviated: GBSSI) (Akasuka and Nelson, J. Biol. Chem., 241, (1966),2280–2285; Shure et al., Cell 35 (1983), 225–233), produces a starchessentially consisting of amylopectin. For potato, genotypes wereproduced both by means of chemical mutagenesis of a haploid line(Hovenkamp-Hermelink et al., Theor. Appl. Genet., 225, (1987), 217–221)and by means of antisense inhibition of the GBSSI-gene, whose starchesessentially consist of amylopectin starch. In comparison with starchesof the corresponding wild type plants, such waxy potato starches do notexhibit any differences with regard to phophate content, the morphologyof the starch granule or the ion content (Visser et al., Starch/Stärke,49, (1997), 438–443).

Furthermore, maize mutants are commercially available which exhibitstarches with amylose contents of about 50% or about 70% (amylosecontent determined by potentiometric determination of the iodine-bindingcapacity) and which are designated Hylon V® or HylonVII® (NationalStarch and Chemical Company, Bridgewater, N.J., U.S.A.). Moreover, alsomaize hybrids have been described which synthesise so-called “lowamylopectin starch” (LAPS) and exhibit a content of high molecular(“high mol weight”) amylopectin of about 2.5% and an amylose content ofabout 90% (potentiometric determination of the iodine-binding capacity)(Shi et al., J. Cereal Science 27, (1998), 289–299).

Furthermore, transgenic potato plants have been described which, due tothe antisense-inhibition of the branching enzyme I (=BEI) and thebranching enzyme II (=BEII) gene, synthesise a potato starch whichexhibits an amylose content of up to 75% by colorimetric determinationof the amylose content according to the method described by Morrison andLaignelet (J. Cereal Sci. 1, (1983), 9–20) (Schwall et al., NatureBiotechn. 18, (2000), 551–554). These potato starches are characterisedby a phosphate content of the starch which is up to 6 times highercompared to corresponding wild type plants. Furthermore, theinternational patent application WO 97/11188 describes transgenic potatoplants which, due to their antisense inhibition of the R1 gene and theBEI gene synthesise a starch with an amylose content of more than 70%,the amylose content having been determined according to the method byHovenkamp & Hermelink (Potato Research 31, (1988), 241–246).

Transgenic potato plant cells and potato plants synthesising a starchhaving an amylose content of more than 75% (calorimetric determinationof the amylose content according to Hovenkamp & Hermelink (PotatoResearch 31, (1988), 241–246) and, at the same time, a reduced phosphatecontent in comparison with corresponding wild type plants have not beendescribed in the state of the art so far. The same applies to the potatostarches that can be isolated from these potato plant cells and plantsand to methods for the production of such starches. However, theprovision of such starches is desirable since their physico-chemicalproperties can be expected to be advantageously useful for variousindustrial applications.

Thus, the technical problem underlying the present invention is toprovide plant cells and plants synthesising starch which has an amylosecontent of more than 75% (calorimetric determination of the amylosecontent according to Hovenkamp & Hermelink (Potato Research 31, (1988),241–246) and a reduced phosphate content in comparison with thephosphate content of starch from corresponding wild type plant cells andplants that have not been genetically modified, as well as to providesuch starch which differs from the starches described in the state ofthe art in its structural and/or functional properties and is, thus,more suitable for general and/or specific purposes.

This technical problem has been solved by providing the embodimentscharacterised in the claims.

SUMMARY OF THE INVENTION

Thus, the present invention relates to a transgenic plant cell which isgenetically modified, wherein the genetic modification leads to areduction of the activity of one or more R1 proteins occurringendogenously in the plant cell and to the reduction of the activity ofone or more BEI proteins occurring endogenously in the plant cell and tothe reduction of the activity of one or more BEII proteins occurringendogenously in the plant cell in comparison with corresponding plantcells of wild type plants, the cells not being genetically modified.

The genetic modification may be any genetic modification leading to areduction of the activity of one or more R1 proteins occurringendogenously in the plant cell and to the reduction of the activity ofone or more BEI proteins occurring endogenously in the plant cell and tothe reduction of the activity of one or more BEII proteins occurringendogenously in the plant cell in comparison with corresponding plantcells of wild type plants, the cells not being genetically modified.

In this context, the term “transgenic” means that the plant cells of theinvention differ in their genetic information from corresponding plantcells which are not genetically modified due to a genetic modification,in particular the introduction of one or more foreign nucleic acidmolecules.

In this context, the term “genetically modified” means that the plantcell is modified in its genetic information due to the introduction ofone or more foreign nucleic acid molecules and that the presence and/orthe expression of the foreign nucleic acid molecule/s lead/s to aphenotypic modification. In this context, phenotypic modificationpreferably relates to a measurable modification of one or more functionsof the cells. Genetically modified plants cells of the invention, forexample, exhibit a reduction of the expression of one or more R1 genesoccurring endogenously in the plant cell and a reduction of theexpression of one or more BEI genes occurring endogenously in the plantcell and a reduction of the expression of one or more BEII genesoccurring endogenously in the plant cell in comparison withcorresponding plant cells of wild type plants, the cells not beinggenetically modified, and/or a reduction of the activity of one or moreR1 proteins occurring endogenously in the plant cell and a reduction ofthe activity of one or more BEI proteins occurring endogenously in theplant cell and a reduction of the activity of one or more BEII proteinsoccurring endogenously in the plant cell in comparison withcorresponding plant cells of wild type plants, the cells not beinggenetically modified.

Within the meaning of the present invention, the term “reduction of theactivity” means a reduction of the expression of endogenous genesencoding R1, BEI and/or BEII proteins and/or a reduction of the amountof R1, BEI and/or BEII proteins in the cells and/or a reduction of theenzymatic activity of the R1, BEI and/or BEII proteins in the cells.

In the context of the present invention, the term “reduction ofexpression” refers to a reduction of the amount of transcripts of therespective endogenous gene in a plant cell of the invention as comparedto a corresponding wild-type plant cell. The reduction of the expressioncan, for instance, be determined by measuring the amount of transcriptsencoding R1, BEI or BEII proteins, e.g. by means of Northern blotanalysis or RT-PCR. In this context, a reduction preferably means areduction of the amount of transcripts in comparison with correspondingcells that have not been genetically modified by at least 50%, inparticular by at least 70%, more preferably by at least 85% and mostpreferably by at least 95%.

The reduction of the amount of R1, BEI and/or BEII proteins can, forinstance, be determined by means of Western blot analysis. In thiscontext, a reduction preferably means a reduction of the amount of R1,BEI and/or BEII proteins in comparison with corresponding cells whichhave not been genetically modified by at least 50%, in particular by atleast 70%, more preferably by at least 85% and most preferably by atleast 95%.

Methods for determining the reduction of the enzymatic activity of theR1, BEI and BEII proteins are known to the person skilled in the art andwill be described further below for each protein individually. In thecontext of the present invention, the term “R1 protein” relates toproteins which have been described, for example, in Lorberth et al.(Nature Biotech. 16, (1998), 473–477) and in the international patentapplications WO 98/27212, WO 00/77229, WO 00/28052 and which have thefollowing characteristics. Important characteristics of R1 proteins arei) their amino acid sequence (see, for example, GenBank Acc. No. A61831,Y09533); ii) their localisation in the plastides of plant cells; iii)their ability to influence the degree of phosphorylation of starch inplants.

Further, the term “R1 protein” refers to a protein catalysing thephosphorylation of starch in dikinase-type reaction in which threesubstrates, an a-polyglucan, ATP and H₂O are converted into threeproducts, an a-polyglucan-P, AMP and orthophosphate (Ritte et al., PNAS99(10) (2002), 7166–7171).

The inhibition of the R1 gene encoding an R1 protein from potato intransgenic potato plants, for example, leads to a reduction of thephosphate content of the starch which can be isolated from the potatotuber. Moreover, Lorberth et al. were able to demonstrate that the R1protein from Solanum tuberosum is able to phosphorylate bacterialglycogen when the corresponding R1 cDNA is expressed in E. coli(Lorberth et al., Nature Biotech. 16, (1998), 473–477).

Ritte et al. (Plant J. 21, (2000), 387–391) were able to show that theR1 protein from Solanum tuberosum in potato plants binds to starchgranules in a reversible way, wherein the strength of the binding to thestarch granule depends on the metabolic status of the plant. In potatoplants, the protein is mainly present in starch granule bound form inleaves that have been kept in the dark. After exposing the leaves tolight, however, the protein is mainly present in the soluble form whichis not bound to the starch granule.

Furthermore, the inhibition of the expression of the R1 gene from potatoin transgenic potato plants or in the tubers thereof leads to areduction of the so-called “cold-induced-sweetenings” (Lorberth et al.,Nature Biotech. 16, (1998), 473–477).

In the context of the present invention, the term “R1 protein” alsorelates to proteins which exhibit a significant homology (identity) ofat least 60%, preferably of at least 80%, more preferably of at least90% to the amino acid sequence stated under SEQ ID NO: 6 or under theGenBank Acc. No. Y09533 or A61831, and which are able to modify thedegree of phosphorylation of polysaccharides such as, for example,starch and/or glycogen. Preferably, the R1 protein originates frompotato (GenBank Acc. No. Y09533 or A61831).

Preferably, an R1 protein, as addressed in the embodiments of thepresent invention, is encoded by a nucleic acid molecule whichhybridises, advantageously under stringent conditions, with the nucleicacid molecule having the nucleotide sequences shown under SEQ ID NO: 5and which encodes a polypeptide having the activity of an R1 protein.

Within the present invention the term “hybridization” meanshybridization under conventional hybridization conditions (also referredto as “low stringency conditions”), preferably under stringentconditions (also referred to as “high stringency conditions”), as forinstance described in Sambrook and Russell (2001), Molecular Cloning, ALaboratory Manual, CSH Press, Cold Spring Harbour, N.Y., U.S.A. Withinan especially preferred meaning the term “hybridization” means thathybridization occurs under the following conditions:

-   Hybridization buffer: 2×SSC; 10× Denhardt solution (Fikoll    400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM    Na₂HPO_(4; 250) μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or    -   0.25 M of sodium phosphate buffer, pH 7.2;    -   1 mM EDTA    -   7% SDS-   Hybridization temperature T=60° C.-   Washing buffer: 2×SSC; 0.1% SDS-   Washing temperature T=60° C.

Nucleic acid molecules which hybridize with a nucleic acid moleculehaving the nucleotide sequence shown under SEQ ID NO: 5 can, inprinciple, encode a R1 protein from any organism expressing such aprotein.

Such hybridizing nucleic acid molecules can for instance be isolatedfrom genomic libraries or cDNA libraries of plants. Alternatively, theycan be prepared by genetic engineering or chemical synthesis.

Such nucleic acid molecules may be identified and isolated with the useof a nucleic acid molecule encoding an R1 protein as disclosed herein orparts of such a molecule or reverse complements of such a molecule, forinstance by hybridization according to standard methods (see forinstance Sambrook and Russell (2001), Molecular Cloning. A LaboratoryManual, CSH Press, Cold Spring Harbor, N.Y., U.S.A).

Nucleic acid molecules possessing the same or substantially the samenucleotide sequence as indicated in SEQ ID NO: 5 or parts thereof can,for instance, be used as hybridization probes. The fragments used ashybridization probes can also be synthetic fragments which are preparedby usual synthesis techniques, and the sequence of which substantiallycoincides with that of a nucleic acid molecule specifically describedherein.

The hybridizing nucleic acid molecules also comprise fragments,derivatives and allelic variants of the nucleic acid molecule having thenucleotide sequence shown under SEQ ID NO: 5. Herein, fragments areunderstood to mean parts of the nucleic acid molecules which are longenough to encode an R1 protein. In this connection, the term derivativemeans that the sequences of these nucleic acid molecules differ from thesequence of an above-described nucleic acid molecule in one or morepositions and show a high degree of homology to such a sequence. In thiscontext, homology means a sequence identity of at least 40%, inparticular an identity of at least 60%, preferably of at least 65%, morepreferably of at least 70%, even more preferably of at least 80%, inparticular of at least 85%, furthermore preferred of at least 90% andparticularly preferred of at least 95%. Most preferably homology means asequence identity of at least n %, wherein n is an integer between 40and 100, i.e. 40≦n≦100. Deviations from the above-described nucleic acidmolecules may have been produced, e.g., by deletion, substitution,insertion and/or recombination.

Preferably, the degree of homology is determined by comparing therespective sequence with the nucleotide sequence of the coding region ofSEQ ID No: 5. When the sequences which are compared do not have the samelength, the degree of homology preferably refers to the percentage ofnucleotide residues in the shorter sequence which are identical tonucleotide residues in the longer sequence. The degree of homology canbe determined conventionally using known computer programs such as theClustalW program (Thompson et al., Nucleic Acids Research 22 (1994),4673–4680) distributed by Julie Thompson (Thompson@EMBL-Heidelberg.DE)and Toby Gibson (Gibson@EMBL-Heidelberg.DE) at the European MolecularBiology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany.ClustalW can also be downloaded from several websites including IGBMC(Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/)and EBI (ftp://ftp.ebi.ac.uk/pub/software/) and all sites with mirrorsto the EBI (European Bioinformatics Institute, Wellcome Trust GenomeCampus, Hinxton, Cambridge CB10 1SD, UK).

When using ClustalW program version 1.8 to determine whether aparticular sequence is, for instance, 90% identical to a referencesequence according to the present invention, the settings are set in thefollowing way for DNA sequence alignments:

-   KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10,    GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

For protein sequence alignments using ClustalW program version 1.8 thesettings are the following: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3,GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET,ENDGAPS(OFF), NOPGAP, NOHGAP.

Homology, moreover, means that there is a functional and/or structuralequivalence between the corresponding nucleic acid molecules or proteinsencoded thereby. Nucleic acid molecules which are homologous to one ofthe above-described molecules and represent derivatives of thesemolecules are generally variations of these molecules which representmodifications having the same biological function. They may be eithernaturally occurring variations, for instance sequences from othermicroorganisms, or mutations, and said mutations may have formednaturally or may have been produced by deliberate mutagenesis.Furthermore, the variations may be synthetically produced sequences. Theallelic variants may, e.g., be naturally occurring variants orsynthetically produced variants or variants produced by recombinant DNAtechniques.

The proteins encoded by the different variants of the nucleic acidmolecule having the nucleotide sequence shown under SEQ ID NO: 5 possesscertain characteristics they have in common. These include for instanceenzymatic activity, molecular weight, immunological reactivity,conformation, etc., and physical properties, such as for instance themigration behavior in gel electrophoreses, chromatographic behavior,sedimentation coefficients, solubility, spectroscopic properties,stability, pH optimum, temperature optimum etc.

In the context of the present invention, the term “R1 gene” relates to anucleic acid molecule (e.g. cDNA, DNA) encoding an “R1 protein” asdescribed above. Nucleic acid molecules encoding R1 proteins have beendescribed for various plants such as, e.g. maize (WO 98/27212 A1), rice(WO 00/28052 A1) and wheat (WO 00/77229 A1). Preferably, the R1 geneoriginates from potato, an R1 cDNA from potato with the nucleotidesequence stated under SEQ ID NO: 5 or GenBank Acc. No. Y09533 or A61831is particularly preferred.

In the context of the present invention, the term “branching enzyme” or“BE protein” (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase, E.C.2.4.1.18) relates to a protein catalysing a transglycosylation reaction,wherein (α-1,4-linkings of an α-1,4-glucan donor are hydrolysed and theα-1,4-glucan chains released thereby are transferred to an α-1,4-glucanacceptor chain and are thereby converted into α-1,6-linkings. Within themeaning of the present invention, the term “BE gene” is a gene encodinga “BE protein”.

In the context of the present invention, the term “BEI protein” relatesto a branching enzyme (=BE) of the isoform I, preferably the BEI proteinoriginates from potato plants.

The designation of the isoforms follows the nomenclature suggested bySmith-White and Preiss (Smith-White and Preiss, Plant Mol. Biol. Rep. 12(1994), 67–71; Larsson et al., Plant Mol. Biol. 37, (1998), 505–511).This nomenclature assumes that all branching enzymes exhibiting a higherhomology (identity) on the amino acid level to the BEI protein frommaize having the amino acid sequence shown under SEQ ID NO: 9 (GenBankAcc. No. D11081; Baba et al., Biochem. Biophys. Res. Commun. 181 (1),(1991), 87–94; Kim et al., Gene 216, (1998), 233–243) than to the BEIIprotein from maize having the amino acid sequence shown under SEQ ID NO:10 (GenBank Acc. No. AF072725, U65948) are designated branching enzymesof the isoform I or, in short, BEI proteins.

In the context of the present invention, the term “BEI gene” relates toa nucleic acid molecule (e.g. cDNA, DNA) encoding a “BEI protein”,preferably a BEI protein from potato plants. Such nucleic acid moleculeshave been described for numerous plants, for example for maize (GenBankAcc. No. D11081, AF 072724), rice (GenBank Acc. No. D11082), pea(GenBank Acc. No. X80010) and potato. Different forms of the BEI gene orthe BEI protein from potato have been described, for example, byKhoshnoodi et al. (Eur. J. Biochem. 242 (1) (1996), 148–155), GenBankAcc. No. Y08786 and by Kossmann et al. (Mol. Gen. Genet. 230 (1991),39–44). In potato plants, the BEI gene is expressed mainly in the tubersand hardly in the leaves (Larsson et al., Plant Mol. Biol. 37, (1998),505–511).

Preferably, a BEI protein, as addressed in the embodiments of thepresent invention, is encoded by a nucleic acid molecule whichhybridises, advantageously under stringent conditions, with the nucleicacid molecule having the nucleotide sequence shown under SEQ ID NO: 7and which encodes a polypeptide having branching enzyme activity.

The definition for the term “hybridisation” as defined above inconnection with R1 proteins applies equally for the definition ofnucleic acid molecules hybridising with the nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO: 7. Preferably, a BEI protein asreferred to herein displays a sequence identity of at least 60%, inparticular of at least 75%, preferably of at least 85%, more preferablyat least 90% and even more preferably at least 95% to the amino acidsequence depicted under SEQ ID NO: 8.

In the context of the present invention, the term “BEII protein” relatesto a branching enzyme (=BE) of the isoform II, preferably it originatesfrom potato plants. Within the meaning of the present invention, allenzymes exhibiting a higher homology (identity) on the amino acid levelto the BEII protein from maize (GenBank Acc. No. AF072725, U65948) thanto the BEI protein from maize (GenBank Acc. No. D 11081, AF 072724) areto be designated BEII protein.

In the context of the present invention, the term “BEII gene” relates toa nucleic acid molecule (e.g. cDNA, DNA) encoding a “BEII protein”,preferably a BEII protein from potato plants. Such nucleic acidmolecules have been described for numerous plants, for example, forpotato (GenBank Acc. No. AJ00004, AJ011888, AJ011889, AJ011885,AJ011890), maize (AF072725, U65948), barley (AF064561), rice (D16201)and wheat (AF286319). In potato plants, the BEII gene is expressedmainly in the leaves and hardly in the tubers (Larsson et al., PlantMol. Biol. 37, (1998), 505–511).

Preferably, a BEII protein, as addressed in the embodiments of thepresent invention, is encoded by a nucleic acid molecule whichhybridises, advantageously under stringent conditions, with the nucleicacid molecule having the nucleotide sequence shown under SEQ ID NO: 9and which encodes a polypeptide having branching enzyme activity.

The definition for the term “hybridisation” as defined above inconnection with R1 proteins applies equally for the definition ofnucleic acid molecules hybridising with the nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO: 3. Preferably, a BEII protein asreferred to herein displays a sequence identity of at least 60%, inparticular of at least 75%, preferably of at least 85%, more preferablyat least 90% and even more preferably at least 95% to the amino acidsequence depicted under SEQ ID NO: 4.

In a preferred embodiment of the present invention, the geneticmodification of the transgenic plant cell of the invention is theintroduction of one or more foreign nucleic acid molecules the presenceand/or expression of which leads to a reduction of the activity of R1and BEI and BEII proteins in comparison with corresponding plant cellsof wild type plants, the cells not being genetically modified.

Preferably, this reduction of activity is achieved by inhibiting theexpression of the endogenous genes encoding R1 proteins, BEI proteinsand BEII proteins.

The production of the plant cells of the invention can be achieved bydifferent methods known to the person skilled in the art, e.g. bymethods leading to an inhibition of the expression of endogenous genesencoding an R1, BEI or BEII protein. These include, for example, theexpression of a corresponding antisense RNA, the provision of moleculesor vectors mediating a co-suppression effect, the expression of aribozyme constructed accordingly which specifically cleaves transcriptsencoding an R1, BEI or BEII protein or the so-called “in-vivomutagenesis”. Furthermore, the reduction of the R1 and/or the BEI and/orthe BEII activity in the plant cells may also be caused by thesimultaneous expression of sense and antisense RNA molecules of thetarget gene to be repressed, preferably of the R1 and/or the BEI and/orthe BEII gene, a technique which is commonly referred to as RNAinterference (RNAi) (Bosher and Labouesse, Nature Cell Biology 2,(2000), E31–E36; Waterhouse et al., PNAS 95, (1998), 13959–13964).Furthermore, by the use of double-stranded RNA molecules comprisingpromoter sequences, a transcriptional inactivation of the promoter canbe achieved. These and further methods for reducing the activity ofproteins will be described in more detail below. All these methods arebased on the introduction of one or more foreign nucleic acid moleculesinto the genome of plant cells.

Within the context of the present invention, the term “foreign nucleicacid molecule” is understood to be a molecule which either does notoccur naturally in corresponding plant cells or which does not occurnaturally in the plant cells in the concrete spatial order or which islocated at a position in the genome of the plant cell at which it doesnot occur naturally. The foreign nucleic acid molecule preferably is arecombinant molecule which consists of various elements the combinationor specific spatial order of which does not occur naturally in plantcells.

The foreign nucleic acid molecule can, for instance, be a so-called“triple construct” which is understood to be a single vector for planttransformation which contains both the genetic information forinhibiting the expression of one or more endogenous R1 genes and forinhibiting the expression of one or more BEI and BEII genes or thepresence or expression of which leads to the reduction of the activityof one or more R1, BEI and BEII proteins.

In another embodiment, the foreign nucleic acid molecule may be aso-called “double construct” which is understood to be a vector forplant transformation which contains the genetic information forinhibiting the expression of two of the three target genes (R1, BEI,BEII gene) or the presence or expression of which leads to the reductionof the activity of two of the three target proteins (R1, BEI, BEIIproteins). In this embodiment of the invention, the inhibition of theexpression of the third target gene and/or the reduction of the activityof the third target protein takes place by means of a separate foreignnucleic acid molecule which contains the corresponding geneticinformation for exerting this inhibiting effect.

In another embodiment of the invention, it is not a triple constructthat is introduced into the genome of the plant cell but severaldifferent foreign nucleic acid molecules, one of these foreign nucleicacid molecules being for example a DNA molecule which, for instance, isa co-suppression construct leading to a reduction of the expression ofone or more endogenous R1 genes, and a further foreign nucleic acidmolecule being a DNA molecule encoding, for example, an antisense RNAleading to a reduction of the expression of one or more endogenous BEIand/or BEII genes. In principle, as regards the construction of theforeign nucleic acid molecules, it is also suitable to use everycombination of antisense, co-suppression and ribozyme constructs orin-vivo mutagenesis, which all lead to a simultaneous reduction of thegene expression of endogenous genes encoding one or more R1, BEI andBEII proteins or which lead to a simultaneous reduction of the activityof one or more R1, BEI and BEII proteins.

In this case, the foreign nucleic acid molecules can be introducedsimultaneously (“co-transformation”) or consecutively, i.e. one afterthe other (“super transformation”), into the genome of the plant cell.

In another embodiment of the invention, at least one antisense RNA isexpressed for reducing the activity of one or more R1 proteins and/orBEI proteins and/or BEII proteins in plant cells.

For inhibiting the gene expression by means of antisense orco-suppression technology, it is possible to use for instance a DNAmolecule which comprises the entire sequence encoding an R1 and/or BEIand/or BEII protein, including flanking sequences that may optionally bepresent, as well as DNA molecules which only comprise parts of thecoding sequence and/or flanking sequences, wherein these parts must belong enough to lead to an antisense effect or a co-suppression effect inthe cells. In general, sequences having a minimum length of 15 bp,preferably a length of 100 to 500 bp, in particular sequences having alength of more than 500 bp, are suitable for an efficient antisense orco-suppression inhibition. Usually, DNA molecules which are shorter than5000 bp, preferably sequences which are shorter than 2500 bp are used.

For antisense or co-suppression approaches, it is also suitable to useDNA sequences which have a high degree of homology to the sequences thatoccur endogenously in the plant cell and that encode R1, BEI or BEIIproteins. The minimum degree of homology should be higher than 65%. Itis preferred to use sequences having a homology of at least 90%, inparticular between 95 and 100%.

Moreover, also introns, i.e. of non-coding regions of genes encoding R1,BEI and/or BEII proteins, are conceivable for use to achieve anantisense or a co-suppressive effect.

The use of intron sequences for inhibiting the gene expression of genesencoding proteins of the starch biosynthesis has been described in theinternational patent applications WO 97/04112, WO 97/04113, WO 98/37213,WO 98/37214.

The person skilled in the art knows how to achieve an antisense and aco-suppressive effect. The method of co-suppression inhibition wasdescribed, for instance, in Jorgensen (Trends Biotechnol. 8 (1990),340–344), Niebel et al. (Curr. Top. Microbiol. Immunol. 197 (1995),91–103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995),43–46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995, 149–159),Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311–317), de Borne et al.(Mol. Gen. Genet. 243 (1994), 613–621).

The skilled persons are also familiar with the expression of ribozymesfor reducing the activity of certain enzymes in cells and is described,for example, in EP-B1 0 321 201. The expression of ribozymes in plantcells has been described, for instance, in Feyter et al. (Mol. Gen.Genet. 250, (1996), 329–338).

Furthermore, the R1 and/or BEI and/or BEII activity in plant cells mayalso be reduced by the so-called “in-vivo mutagenesis” in which a hybridRNA-DNA oligonucleotide (“chimeroplast”) is introduced into cells bymeans of the transformation of cells (Kipp, P. B. et al., Poster at the5^(th) International Congress of Plant Molecular Biology, 21–27September1997, Singapore; R. A. Dixon and C. J. Arntzen, Meeting report to“Metabolic Engineering in Transgenic Plants”, Keystone Symposia, CopperMountain, Colo., U.S.A., TIBTECH 15, (1997), 441–447; internationalpatent application WO 95/15972; Kren et al., Hepatology 25, (1997),1462–1468; Cole-Strauss et al., Science 273, (1996), 1386–1389).

A part of the DNA component of the RNA-DNA oligonucleotide is homologousto a nucleic acid sequence of an endogenous R1, BEI and/or BEII gene, ithas, however, a mutation in comparison with an endogenous R1, BEI and/orBEII gene or it contains a heterologous region which is embraced by thehomologous regions.

It is possible to transfer the mutation or heterologous region containedin the DNA component of the RNA-DNA molecule into the genome of a plantcell by means of base pairing of the homologous regions of the RNA-DNAoligonucleotide and the endogenous nucleic acid molecule, followed byhomologous recombination. As a result, the activity of one or more R1,BEI and/or BEII proteins is reduced.

In addition, the reduction of the R1 and/or BEI and/or BEII activity inthe plant cells may also be caused by the simultaneous expression ofsense and antisense RNA molecules of the target gene that is to berepressed, preferably of the R1 and/or the BEI and/or the BEII gene.

This can be achieved, for instance, by using chimeric constructs whichcontain inverted repeats of the respective target gene or of parts ofthe target gene. In this case, the chimeric constructs encode sense andantisense RNA molecules of the respective target gene. Sense andantisense RNA are synthesised simultaneously in planta as one RNAmolecule, wherein the sense and the antisense RNA may be separated fromeach other by a spacer and form a double-stranded RNA-molecule. It waspossible to show that the introduction of inverted repeat DNA constructsinto the genome of plants is a very efficient method for repressing thegenes corresponding to the inverted repeat DNA constructs (Waterhouse etal., Proc. Natl. Acad. Sci. USA 95, (1998), 13959–13964; Wang andWaterhouse, Plant Mol. Biol. 43, (2000), 67–82; Singh et al.,Biochemical Society Transactions vol. 28, part 6 (2000), 925–927; Liu etal., Biochemical Society Transactions vol. 28, part 6 (2000); 927–929;Smith et al., Nature 407, (2000), 319–320; international patentapplication WO 99/53050 A1). Sense and antisense sequences of the targetgene or of the target genes can also be expressed separately using thesame or different promoters (Nap, J.-P. et al., 6^(th) InternationalCongress of Plant Molecular Biology, Quebec, 18–24 June 2000; PosterS7–27, lecture session S7).

Thus, it is also possible to reduce the R1 and/or BEI and/or BEIIactivity in the plant cells by the production of double-stranded RNAmolecules of R1 and/or BEI and/or BEII genes. Preferably, invertedrepeats of DNA molecules of R1 and/or BEI and/or BEII genes or cDNAs areintroduced into the genome of plants for this purpose, wherein the DNAmolecules to be transcribed (R1, BEI or BEII gene or cDNA or fragmentsof these genes or cDNAs) are under the control of a promoter whichcontrols the expression of said DNA molecules.

Furthermore, it is known that in planta the formation of double-strandedRNA molecules of promoter DNA molecules in plants can lead in trans to amethylation and a transcriptional inactivation of homologous copies ofthese promoters which are called target promoters in the following(Mette et al., EMBO J. 19, (2000), 5194–5201).

Therefore, it is possible to reduce the gene expression of a certaintarget gene (e.g. R1, BEI or BEII gene) by means of the inactivation ofthe target promoter, the target gene being naturally controlled by thistarget promoter.

This means that, in this case, in contrast to the original function ofpromoters in plants, the DNA molecules which comprise the targetpromoters of the genes to be repressed (target genes) are not used ascontrol elements for the expression of genes or cDNAs but astranscribable DNA molecules themselves.

For producing the double-stranded target promoter RNA molecules inplanta which may be present there as RNA hairpin molecules, constructsare preferred to be used which contain inverted repeats of the targetpromoter DNA molecule, the target promoter DNA molecules being under thecontrol of a promoter which controls the gene expression of said targetpromoter DNA molecules.

Then, these constructs are introduced into the genome of plants. Theexpression of the inverted repeats of said target promoter DNA moleculesleads to the formation of double-stranded target promoter RNA moleculesin planta (Mette at el., EMBO J. 19, (2000), 5194–5201). In this way, itis possible to inactivate the target promoter.

Therefore, the R1 and/or BEI and/or BEII activity in the plant cells canalso be reduced by generating double-stranded RNA molecules of promotersequences of R1 and/or BEI and/or BEII genes. For this purpose, invertedrepeats of promoter DNA molecules of R1 and/or BEI and/or BEII promotersare preferred to be introduced into the genome of plants, the targetpromoter DNA molecules to be transcribed (R1, BEI and/or BEII promoter)being under the control of a promoter which controls the expression ofsaid target promoter DNA molecules. The promoter sequences from R1and/orBEI and/or BEII genes necessary for carrying out the present embodimentcan be provided by methods known to the skilled person and described inthe literature such as in Sambrook and Russell (2001), MolecularCloning, CSH Press, Cold Spring Harbor, N.Y., U.S.A. The methods may forexample include the preparation of a genomic library from the plant inwhich the activity of R1, BEI and BEII proteins shall be reduced,screening of the library for clones containing the sequence flanking thecoding region of the respective gene in 5′-direction by the help of aprobe comprising a coding sequence for the R1 or BEI or BEII protein asdescribed above and finally sequencing positive clones by conventionaltechniques.

Moreover, the skilled person knows that the reduction of activity of oneor more R1, BEI and/or BEII proteins can be achieved by means of theexpression of non-functional derivatives, in particular trans-dominantmutants of such proteins, and/or by means of the expression ofantagonists/inhibitors of such proteins.

Antagonists/inhibitors of such proteins comprise, for instance,antibodies, antibody fragments or molecules having similar bindingproperties. A cytoplasmatic scFv antibody, for example, was used formodulating the activity of the phytochrome A protein in geneticallymodified tobacco plants (Owen, Bio/Technology 10 (1992), 790–4; Review:Franken, E., Teuschel, U. and Hain, R., Current Opinion in Biotechnology8, (1997), 411–416; Whitelam, Trends Plant Sci. 1 (1996), 268–272).

Therefore, a plant cell of the invention is also subject matter of thepresent invention, wherein said foreign nucleic acid molecule thepresence and/or expression of which causes a reduction of R1, BEI andBEII activity in said plant cell is selected from the group consistingof

-   a) DNA molecules which encode at least one antisense RNA leading to    a reduction of the expression of endogenous genes encoding R1    proteins and/or BEI proteins and/or the BEII proteins, preferably    encoding R1, BEI and BEII proteins;-   b) DNA molecules which, through a co-suppression effect, lead to a    reduction of the expression of endogenous genes encoding R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins;-   c) DNA molecules encoding at least one ribozyme which specifically    cleaves transcripts of endogenous genes encoding R1 proteins and/or    BEI proteins and/or BEII proteins, preferably encoding R1, BEI and    BEII proteins;-   d) nucleic acid molecules which have been introduced by means of    in-vivo mutagenesis and which lead to a mutation or an insertion of    a heterologous sequence in the genes encoding endogenous R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins, wherein the mutation or insertion leads to a    reduction of the expression of genes encoding R1 proteins and/or BEI    proteins and/or BEII proteins, or the synthesis of inactive R1    and/or BEI and/or BEII proteins; and-   e) DNA molecules which simultaneously encode at least one antisense    RNA and at least one sense RNA, wherein said antisense RNA and said    sense RNA form a double-stranded RNA molecule which leads to a    reduction of the expression of endogenous genes encoding R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins.

In another embodiment, the present invention relates to transgenic plantcells which synthesises a modified starch. The transgenic plant cells ofthe invention synthesise a modified starch which is modified in itsphysico-chemical properties, in particular the amylose/amylopectinratio, the phosphate content, the viscosity behaviour, the size of thestarch granules and/or the form of the starch granules in comparisonwith starch synthesised in wild type plants so that it is more suitablefor specific purposes of application.

It was surprisingly found that the composition of the starch is modifiedin the plant cells of the invention in such a way that it has an amylosecontent of at least 75% and a reduced phosphate content in comparisonwith starch from plant cells from corresponding wild type plants, sothat said starch is more suitable for specific purposes of application.

A plant cell of the invention which contains modified starch having anamylose content of at least 75% and a reduced phosphate content comparedto the starch of corresponding plant cells of wild type plants, thecells not being genetically modified, is also subject matter of thepresent invention.

In the context of the present invention, the amylose content isdetermined according to the method by Hovenkamp-Hermelink et al.described below in connection with potato starch (Potato Research 31,(1988), 241–246). This method can also be used for isolated starches ofother plant species. The person skilled in the art is familiar withmethods for isolating starches.

Within the meaning of the present invention, the term “phosphatecontent” relates to the content of phosphate bound covalently in form ofstarch phosphate monoesters.

In the context of the present invention, the expression “reducedphosphate content” means that the overall phosphate content of phosphatecovalently bound and/or the phosphate content in the C-6 position of thestarch synthesised in the plant cells of the invention is reduced by atleast 20%, preferably by at least 50%, more preferably by at least 80%in comparison with starch from plant cells of corresponding wild typeplants, the cells not being genetically modified.

Within the meaning of the present invention, the term “phosphate contentin the C-6 position” is understood to be the content of phosphate groupswhich are bound to the carbon atom position “6” of the glucose monomersof the starch. In principle, the positions C2, C3 and C6 of the glucoseunits may be phosphorylated in the starch in vivo. In connection withthe present invention, the phosphate content in the C-6 position (=C6-Pcontent) can be determined through the determination of theglucose-6-phosphate by means of an optic-enzymatic test (Nielsen et al.,Plant Physiol. 105, (1994), 111–117) (see below).

In the context of the present invention, the expression “overallphosphate content” of the starch is understood to be the content ofphosphate bound covalently in form of starch phosphate monoesters in theC2, C3 and C6 position of the glucose units. According to the invention,the content of phosphorylated non-glucans such as, e.g. phospholipids,is not included in the term “overall phosphate content”. Thus,phosphorylated non-glucans must be separated quantitatively beforedetermining the overall phosphate content. The skilled person knowsmethods for separating the phosphorylated non-glucans (e.g.phospholipids) from the starch. Methods for determining the overallphosphate content are known to the person skilled in the art and aredescribed below.

In a preferred embodiment of the invention, the plant cells of theinvention synthesise a starch which has a phosphate content in the C-6position of the glucose monomers of up to 15 nmol C6-P mg⁻¹ starch, inparticular of up to 10 nmol C6-P mg⁻¹ starch, preferably of up to 7 nmolC6-P mg⁻¹ starch, more preferably of up to 4 nmol C6-P mg⁻¹ starch.

In another embodiment, the present invention therefore relates to plantcells according to the invention which synthesise a modified starch,wherein the modified starch is characterised in that it has a modifieddistribution of the side chains. It has been shown that the starchmodified in the plant cells of the invention is characterised not onlyby an increased amylose content and a reduced phosphate content comparedto the starch of corresponding wild type plants, but also by a modifieddistribution of the side chains.

In this embodiment, the term “modified distribution of the side chains”is understood to be an increase in the proportion of short side chainshaving a DP of 26 to 31 by at least 50%, preferably by at least 100%,more preferably by at least 150% and especially preferred by at least200% in comparison with the proportion of short side chains having a DPof 26 to 31 of amylopectin from wild type plants. Moreover, the term“modified distribution of the side chains” means an increase of theproportion of short side chains having a DP of 26 to 31, wherein theincrease of the proportion of short side chains having a DP of 26 to 31is not higher than 800%, in particular not higher than 500% compared tothe proportion of short side chains having a DP of 26 to 31 ofamylopectin from wild type plants. The quantity “DP” means the degree ofpolymerisation.

The proportion of short side chains is determined by the determinationof the proportion in percent that a certain side chain has in theoverall proportion of all side chains. The overall proportion of allside chains is determined through the determination of the overallheight of the peaks which represent the polymerisation degrees of DP 6to 40 in the HPLC chromatogram. The proportion in percent that a certainside chain has in the overall proportion of all side chains isdetermined by the determination of the ratio of the height of the peakrepresenting this side chain in the HPLC chromatogram to the overallheight. The program Chromeleon 6.20 by Dionex, USA can, for instance, beused for determining the peak areas.

In another preferred embodiment, the present invention relates to plantscells of the invention which synthesise a modified starch which form agel after pasting in a 60% (w/v) CaCl₂ solution, the gel having anincreased gel strength compared to the gel from starch of correspondingwild type plant cells that have not been genetically modified.

Within the meaning of the present invention, the term “increased gelstrength” means an increase in the gel strength by at least 1000%, inparticular by at least 2500%, preferably by at least 5000% and morepreferably by at least 10,000%, by 40,000% at the most or by 30,000% atthe most in comparison with the gel strength of starch of correspondingwild type plant cells that have not been genetically modified.

In the context of the present invention, the gel strength is to bedetermined by means of a texture analyzer under the conditions describedbelow. In this case, the pasting of the starch is achieved in an aqueous60% (w/v) CaCl₂ solution since in a purely aqueous system, it is notpossible to achieve pasting of the starch at normal pressure.

In a further preferred embodiment, the present invention relates toplant cells of the invention which, in addition to the aforementionedproperties, the starch of which has a modified morphology of the starchgranules.

In comparison with high-amylose starches which are known so far, inparticular with high-amylose potato starches, the starches of the plantcells of the invention are not only modified in the amylose content, thephosphate content, the distribution of the side chains, the viscositybehaviour and the gel formation behaviour, but also in a modifiedmorphology of the starch granules, which renders these starches moresuitable for certain purposes of application.

These starches, in particular the potato starches, could, for instance,be used instead of rice starches since, after mechanical fragmentation,the starches of the invention have an average size of the starchgranules which is similar to that of rice starches. Compared to ricestarches, the starches of the invention, in particular the potatostarches, however have the advantage that they can be sedimented tolarger units having the form of a bunch of grapes (cf. Example 2) assmall starch granules form bunch-of-grapes-like agglomerations, whichmay be of advantage in the extraction and processing of the starch andby which the costs may be reduced.

Preferably, the morphology of the starch granules contained in the plantcells of the invention is characterised by an agglomeration of smallstarch granules having the form of a bunch of grapes.

In a preferred embodiment, the starches contained in the plant cells ofthe invention are characterised in that the average granule size isreduced compared to the average granule size of corresponding cells ofwild type plants which are not genetically modified.

In the context of the present invention, the term “average granule size”means the granule size which can be determined using, for instance, aphoto sedimentometer of the type “Lumosed FS1” by Retsch GmbH (seebelow).

In a further preferred embodiment of the invention, a reduced averagegranule size is a reduction of the average granule size by at least 20%,preferably by at least 40% and more preferably by at least 60%.

In another preferred embodiment, the starches of the plant cells of theinvention are characterised by an average granule size of less than 20μm, in particular of less than 18 μm, preferably of less than 16 μm andmore preferably of 10–15 μm.

In another preferred embodiment of the invention, the starches of theplant cells of the invention are characterised in that the proportion ofgranules having an average granule size of less than 20 μm is at least70%, preferably at least 75% and more preferably at least 80%.

After mechanical fragmentation of the starch, which may be carried outas described below, the starches of the plant cells of the inventionhave a proportion of granules having a granule size of less than 20 μmof at least 80%, preferably of at least 90% and more preferably of atleast 95%.

A plurality of techniques is available for introducing DNA into a planthost cell. These techniques comprise the transformation of plant cellswith T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenesas a transformation means, the fusion of protoplasts, injection, theelectroporation of DNA, the introduction of DNA by means of thebiolistic approach, and other possibilities.

The use of the Agrobacteria-mediated transformation of plant cells hasbeen researched into intensively and described sufficiently in EP120516;Hoekema, in: The Binary Plant Vector System, Offsetdrukkerij KantersB.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. PlantSci. 4, 1–46 and An et al. EMBO J. 4, (1985), 277–287. For thetransformation of potato, see, for example, Rocha-Sosa et al. (EMBO J.8, (1989), 29–33.).

The transformation of monocotyledonous plants by means ofAgrobacterium-based vectors has also been described (Chan et al., PlantMol. Biol. 22, (1993), 491–506; Hiei et al., Plant J. 6, (1994) 271–282;Deng et al., Science in China 33, (1990), 28–34; Wilmink et al., PlantCell Reports 11, (1992), 76–80; May et al., Bio/Technology 13, (1995),486–492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550–555;Ritchie et al., Transgenic Res. 2, (1993), 252–265). An alternativesystem for the transformation of monocotyledonous plants is thetransformation by the biolistic approach (Wan and Lemaux, Plant Physiol.104, (1994), 37–48; Vasil et al., Bio/Technology 11 (1993), 1553–1558;Ritala et al., Plant Mol. Biol. 24, (1994), 317–325; Spencer et al.,Theor. Appl. Genet. 79, (1990), 625–631), protoplast transformation, theelectroporation of partially permeabilised cells, and the introductionof DNA by means of glass fibers. The transformation of maize, inparticular, has been described repeatedly in the literature (cf., forexample, WO 95/06128, EP0513849, EP0465875, EP0292435; Fromm et al.,Biotechnology 8, (1990), 833–844; Gordon-Kamm et al., Plant Cell 2,(1990), 603–618; Koziel et al., Biotechnology 11 (1993), 194–200; Morocet al., Theor. Appl. Genet. 80, (1990), 721–726).

The successful transformation of other cereal species has also beendescribed, for example in the case of barley (Wan and Lemaux, see above;Ritala et al., see above; Krens et al., Nature 296, (1982), 72–74) andwheat (Nehra et al., Plant J. 5, (1994), 285–297). For the expression ofthe foreign nucleic acid molecule (foreign nucleic acid molecules), inprinciple, any promoter which is active in plant cells can be used. Thepromoter can be chosen in such a way that expression in the plantsaccording to the invention is constitutive, or only in a particulartissue, at a particular point in time of plant development, or at apoint in time determined by external factors. With respect to the plant,the promoter may be homologous or heterologous. Examples of suitablepromoters are the promoter of the cauliflower mosaic virus 35S RNA andthe ubiquitin promoter from maize for constitutive expression, thepatatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23–29),the MCPI promoter of the metallocarboypeptidase inhibitor gene frompotato (Hungarian patent application HU9801674) or the GBSSI promoterfrom potato (international patent application WO 92/11376) fortuber-specific expression in potatoes, or a promoter which ensuresexpression only in photosynthetically active tissues, for example theST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987),7943–7947; Stockhaus et al., EMBO J. 8 (1989), 2445–2451), the Ca/bpromoter (see, for example, U.S. Pat. No. 5,656,496, U.S. Pat. No.5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89, (1992),3654–3658) and the rubisco SSU promoter (see, for example, U.S. Pat. No.5,034,322, U.S. Pat. No. 4,962,028), or the glutelin promoter for anendosperm-specific expression (Leisy et al., Plant Mol. Biol. 14 (1990),41–50; Zheng et al., Plant J. 4, (1993), 357–366; Yoshihara et al., FEBSLett. 383, (1996), 213–218), the shrunken-1 promoter (Werr et al., EMBOJ. 4, (1985), 1373–1380), the HMG promoter from wheat, the USP promoter,the phaseolin promoter, or promoters of maize zein genes (Pedersen etal., Cell 29, (1982), 1015–1026; Quatroccio et al., Plant Mol. Biol. 15(1990), 81–93). The expression of the foreign nucleic acid molecule (theforeign nucleic acid molecules) is of particular advantage in organs ofthe plant that store starch. Such organs are, e.g., the tuber of thepotato plant or the kernels or the endosperm of maize, wheat or riceplants. Thus, promoters mediating the expression in these organs arepreferred to be used.

However, it is also possible to use promoters which are only activatedat a point in time which is determined by external factors (see, forexample, WO 93/07279). Promoters of heat shock proteins, which permitsimple induction, may be of particular interest in this context.Furthermore, seed-specific promoters such as, for example, the Viciafaba USP promoter which ensures seed-specific expression in Vicia fabaand other plants (Fiedler et al., Plant Mol. Biol. 22, (1993), 669–679;Bäumlein et al., Mol. Gen. Genet. 225, (1991), 459–467) can be used.Other promoters which can be employed are fruit-specific promoters asdescribed, for example, in WO 91/01373. A termination sequence whichserves for the correct termination of the transcription and for adding apoly-A tail to the transcript, which is understood to have a function instabilising the transcripts, may furthermore be present. Such elementshave been described in the literature (cf., for example, Gielen et al.,EMBO J. 8 (1989), 23–29) and are freely exchangeable.

The plant cells according to the invention may belong to any plantspecies, i.e. to monocotyledonous or dicotyledonous plants. They arepreferably plant cells from agriculturally useful plants, i.e. plantswhich are grown by man for the purposes of nutrition or for technical,in particular industrial, purposes. The invention preferably relates tofibre-forming (for example flax, hemp, cotton), oil-storing (for examplerape, sunflower, soy bean), sugar-storing (for example sugar beet, sugarcane, sugar millet) and protein-storing plants (for example leguminousplants). In a further preferred embodiment, the invention relates tofodder plants, in particular forage grass and pasture grass (alfalfa,clover, etc.) and vegetable plants (for example tomato, lettuce,chicory).

In another preferred embodiment, the invention relates to plant cellsfrom starch-storing plants (for example wheat, barley, oat, rye, potato,maize, rice, pea, cassava), particularly preferred are plant cells frompotato.

The plant cells of the invention can be used for regenerating wholeplants.

The plants obtainable by regenerating the transgenic plant cells of theinvention are also subject matter of the present invention.

Furthermore, plants which contain the transgenic plant cells of theinvention are also subject matter of the invention.

The transgenic plants may, in principle be plants belonging to any plantspecies, i.e. both monocotyledonous and dicotyledonous plants. They arepreferably useful plants, i.e. plants which are grown by man for thepurposes of nutrition or for technical, in particular industrial,purposes. The invention preferably relates to plant cells offibre-forming (for example flax, hemp, cotton), oil-storing (for examplerape, sunflower, soy bean), sugar-storing (for example sugar beet, sugarcane, sugar millet) and protein-storing plants (for example leguminousplants). In a further preferred embodiment, the invention relates tofodder plants, in particular forage grass and pasture grass (alfalfa,clover, etc.) and vegetable plants (for example tomato, lettuce,chicory).

In another preferred embodiment, the invention relates to starch-storingplants (for example wheat, barley, oat, rye, potato, maize, rice, pea,cassava), particularly preferred are potato plants.

The present invention also relates to a method for the production of atransgenic plant cell which synthesises a modified starch, wherein aplant cell is genetically modified by introducing one or more foreignnucleic acid molecules, the presence and/or expression of which leads toa reduction of the activity of R1, BEI and BEII proteins compared tocorresponding plants cells of wild type plants, the cells not beinggenetically modified.

In a preferred embodiment of the method of the invention, the modifiedstarch is characterised in that it has an amylose content of at least75% and a reduced phosphate content in comparison with starch fromcorresponding wild type plants which are not genetically modified.

The present invention also relates to a method for producing atransgenic plant which synthesises modified starch, wherein

-   a) a plant cell is genetically modified by introducing one or more    foreign nucleic acid molecules the presence and/or expression of    which leads to a reduction of the activity of R1, BEI and BEII    proteins compared to corresponding plant cells of wild type plants,    the cells not being genetically modified;-   b) a plant is regenerated from the cell produced according to a);    and-   c) optionally further plants are produced from the plant produced    according to step b).

In a preferred embodiment of the method of the invention, the modifiedstarch is characterised in that it has an amylose content of at least75% and a reduced phosphate content compared to the starch fromcorresponding wild type plants which are not genetically modified.

In another embodiment of the method of the invention, the modifiedstarches moreover have a modified distribution of the side chains and/ora modified morphology of the starch granules and/or a reduced averagesize of the starch granules and/or form a gel after pasting in anaqueous 60% (w/v) CaCl₂ solution, the gel having an increased gelstrength in comparison with a gel of starch from corresponding wild typeplants which are not genetically modified. The same as has already beensaid above in connection with the plant cells of the invention alsoapplies to the genetic modification introduced according to step a).Regeneration, of plants according to step b) can be made using methodsknown to the person skilled in the art.

Further plants of the methods of the invention can be produced accordingto step c) by means of vegetative propagation (for example usingcuttings, tubers or by means of callus culture and regeneration of wholeplants) or by generative propagation. Generative propagation ispreferably done under controlled conditions, i.e. selected plants havingspecific properties are crossed with each other and propagated. Theperson skilled in the art obviously knows that, for producing the plantcells and plants of the invention, also transgenic plants can be used inwhich the activity of one or two of the aforementioned proteins hasalready been reduced and which, according to the method of theinvention, only have to be genetically modified in such a way that theactivity of the second or third protein is also reduced.

In addition, the skilled person knows that the aforementionedsuper-transformation is not necessarily carried out with primarytransformants but preferably with pre-selected stable transgenic plantswhich advantageously have already been tested for, e.g. fertility,stable expression of the foreign gene, hemizygosity and heterozygosity,etc. in corresponding experiments.

The present invention also relates to the plants obtainable by themethods of the invention.

The present invention also relates to propagation material of plants ofthe invention containing plant cells of the invention as well as of theplants produced according to the methods of the invention. Within themeaning of the present invention, the term “propagation material”comprises parts of the plant which are suitable for producing progeny bythe vegetative or generative route. Examples which are suitable forvegetative propagation are cuttings, callus cultures, rhizomes ortubers. Other propagation material encompasses, for example, fruits,seeds, seedlings, protoplasts, cell cultures and the like. Thepropagation material is preferably seeds.

Furthermore, the present invention relates to the use of one or moreforeign nucleic acid molecules encoding proteins having the enzymaticactivity of R1, BEI and BEII proteins and to the use of fragments ofsaid foreign nucleic acid molecules for producing plant cells or plantsof the invention synthesising a modified starch.

In another embodiment of the invention, the plant cells of the inventionsynthesise a modified starch due to the use according to the inventionof one or more foreign nucleic acid molecules, the modified starch beingcharacterised in that it has an amylose content of at least 75% and/or areduced phosphate content compared to the starch of corresponding wildtype plants which have not been genetically modified and/or a modifieddistribution of the side chains and/or a modified morphology of thestarch granules and/or a reduced average size of the starch granulesand/or a modified starch which forms a gel after pasting in an aqueous60% (w/v) CaCl₂ solution, the gel having an increased gel strength incomparison with a gel of starch from corresponding wild type plant cellswhich are not genetically modified.

In another embodiment, the present invention relates to the use of oneor more foreign nucleic acid molecules for producing plants of theinvention, wherein the foreign nucleic acid molecule is a molecule, orthe foreign nucleic acid molecules are several molecules selected fromthe group consisting of

-   a) DNA molecules which encode at least one antisense RNA leading to    a reduction of the expression of endogenous genes encoding the R1    proteins and/or BEI proteins and/or the BEII proteins, preferably    encoding R1, BEI and BEII proteins;-   b) DNA molecules which, through a co-suppression effect, lead to the    reduction of the expression of endogenous genes encoding R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins;-   c) DNA molecules encoding at least one ribozyme which specifically    cleaves transcripts of endogenous genes encoding R1 proteins and/or    BEI proteins and/or BEII proteins, preferably encoding R1, BEI and    BEII proteins;-   d) nucleic acid molecules which have been introduced by means of    in-vivo mutagenesis and which lead to a mutation or an insertion of    a heterologous sequence in the genes encoding endogenous R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins, wherein the mutation or insertion leads to a    reduction of the expression of the genes encoding R1 proteins and/or    BEI proteins and/or BEII proteins, or the synthesis of inactive R1    and/or BEI and/or BEII proteins; and-   e) DNA molecules which simultaneously encode at least one antisense    RNA and at least one sense RNA, wherein said antisense RNA and said    sense RNA form a double-stranded RNA molecule which leads to a    reduction of the expression of endogenous genes encoding R1 proteins    and/or BEI proteins and/or BEII proteins, preferably encoding R1,    BEI and BEII proteins.

As has already been explained before, the foreign nucleic acid moleculescan be introduced simultaneously or consecutively, i.e. one after theother, into the genome of the plant cell. The simultaneous introductionof the foreign nucleic acid molecules saves time and costs, i.e. theco-transformation in which, preferably in one transformation experimentaccording to the aforementioned methods of the invention, foreignnucleic acid molecules are introduced into the plant cell, the presenceand optionally the expression of which lead to the reduction of theactivity of one or more R1 proteins occurring endogenously in the plantcell and to the reduction of the activity of one or more BEI proteinsoccurring endogenously in the plant cell and to the reduction of theactivity of one or more BEII proteins occurring endogenously in theplant cell in comparison with corresponding plant cells of wild typeplants, the cells not being genetically modified.

Thus, the present invention also relates to compositions containing atleast one of the foreign nucleic acid molecules defined according to theinvention, these foreign nucleic acid molecules being suitable forproducing the transgenic plant cells and/or the transgenic plants of theinvention. Preferably, the presence and/or expression of these foreignnucleic acid molecules in plant cells leads to the reduction of theactivity of R1 and BEI and BEII proteins compared to corresponding plantcells of wild type plants, the cells not being genetically modified.

In this case, in the composition of the invention, the nucleic acidmolecules the presence and/or expression of which in the plant celland/or the plant leads to the reduction of the activity of R1 and BEIand BEII proteins compared to corresponding plant cells of wild typeplants, the cells not being genetically modified, can be containedeither separately or together in one recombinant nucleic acid molecule.In the former case, the composition of the invention can, for instance,contain two or more recombinant nucleic acid molecules and/or vectorsthe joint presence of which in the plant cell leads to said phenotype.In the latter case, a recombinant nucleic acid molecule contains thegenetic information leading to the reduction of the activity of R1 andBEI and BEII proteins compared to corresponding plant cells of wild typeplants, the cells not being genetically modified.

In such a recombinant molecule, for instance, the above-describedforeign nucleic acid molecules the presence and/or expression of whichin a plant cell leads to the reduction of the activity of R1 and BEI andBEII proteins compared to corresponding plant cells of wild type plants,the cells not being genetically modified, can be present as one chimericgene or as separate genes. Examples of such double or multipleconstructs have been described numerously in the literature.

The aforementioned recombinant nucleic acid molecules can be present inany host cell.

In another embodiment, the present invention therefore also relates to ahost cell, in particular a plant cell, containing a composition of theinvention.

The plant cells and plants of the invention synthesise a starch, inparticular in their starch-storing organs, which is modified in itsphysico-chemical properties, in particular the phosphate content and/orthe amylose content, preferably the phosphate content and the amylosecontent, and/or the distribution of the side chains and/or the viscositybehaviour and/or the morphology of the starch granules and/or theaverage size of the starch granules in comparison with starchsynthesised in wild type plants.

Thus, starch which is obtainable from the plant cells, plants and/orpropagation material of the invention is also subject matter of theinvention.

In a preferred embodiment, the starch of the invention is characterisedin that it has an amylose content of at least 75% and a reducedphosphate content in comparison with starch from corresponding wild typeplants which are not genetically modified.

The meaning of the term “increased gel strength” has already beendefined in connection with the description of the plant cells of theinvention.

In comparison with high-amylose starches which are known so far, inparticular with high-amylose potato starches, the starches of theinvention are not only modified in the amylose content, the phosphatecontent, the distribution of the side chains, the viscosity behaviourand the gel formation behaviour, but also in a modified morphology ofthe starch granules, which renders these starches more suitable forcertain purposes of application.

The starches of the invention, in particular the potato starches, could,for instance, be used instead of rice starches since, after mechanicalfragmentation, the starches of the invention have an average size of thestarch granules which is similar to that of rice starches. Compared torice starches, the starches of the invention, in particular the potatostarches, however have the advantage that they can be sedimented tolarger units having the form of a bunch of grapes (cf. Example 2) assmall starch granules form bunch-of-grapes-like agglomerations, whichmay be of advantage in the extraction and processing of the starch andby which the costs may be reduced.

Preferably, morphology of the starch granules of the starch of theinvention is characterised by an agglomeration of small starch granuleshaving the form of a bunch of grapes.

In another embodiment, the starches of the invention are characterisedin that the average granule size is reduced compared to the averagegranule size of corresponding wild type plants which are not geneticallymodified.

In the context of the present invention, the term “average granule size”means the granule size which can be determined using, for instance, aphoto sedimentometer of the type “Lumosed FS1” by Retsch GmbH (seebelow).

In another embodiment of the invention, a reduced average granule sizeis a reduction of the average granule size by at least 20%, preferablyby at least 40% and more preferably by at least 60%.

In another embodiment, the starches of the invention are characterisedby an average granule size of less than 20 μm, in particular of lessthan 18 μm, preferably of less than 16 μm and more preferably of10–15μm.

In another embodiment of the invention, the starches of the inventionare characterised in that the proportion of granules having an averagegranule size of less than 20 μm is at least 70%, preferably at least 75%and more preferably at least 80%.

After mechanical fragmentation of the starch, which may be carried outas described below, the starches of the invention have a proportion ofgranules having a granule size of less than 20 μm of at least 80%,preferably of at least 90% and more preferably of at least 95%.

In a particularly preferred embodiment, the starch of the invention is apotato starch. Moreover, the present invention relates to a method forproducing the starches of the invention comprising the step ofextracting the starch from a plant (cell) of the invention and/or fromstarch-storing parts of such a plant.

Preferably, such a method also comprises the step of harvesting thecultivated plants and/or starch-storing parts of said plants prior toextracting the starch and, particularly preferably, the step ofcultivating the plants of the invention prior to the harvesting.

The person skilled in the art knows methods for extracting the starchfrom plants or from starch-storing parts of plants. Furthermore, methodsfor extracting the starch from various starch-storing plants have beendescribed, e.g. in “Starch: Chemistry and Technology (editors.:Whistler, BeMiller and Paschall (1994), 2^(nd)edition, Academic PressInc. London Ltd.; ISBN 0-12-746270-8; cf., e.g. chapter XII, page412–468: maize and sorghum starches: production; by Watson; ChapterXIII, page 469–479: tapioca, arrowroot and sago starches: production; byCorbishley and Miller; Chapter XIV, page 479–490: potato starch:production and uses; by Mitch; Chapter XV, page 491 to 506: wheatstarch: production, modification and uses; by Knight and Oson; andChapter XVI, page 507 to 528: rice starch: production and uses; byRohmer and Klem; maize starch: Eckhoff et al., Cereal Chem. 73 (1996)54–57), the extraction of maize starch on an industrial scale isgenerally achieved by wet milling. Apparatuses usually used in processesfor extracting starch from plant materials are separators, decanters,hydrocyclones, spray dryers and fluidized-bed dryers.

Moreover, starch which is obtainable using the aforementioned method ofthe invention is also subject matter of the invention.

The starches according to the invention can be modified afterwards byprocesses known to the skilled person and are suitable, in theirunmodified or modified forms, known to the skilled person and aresuitable, in their unmodified or modified forms, for a variety ofapplications in the food or non-food sector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the expression vector ME 5/6 asdescribed further below.

FIG. 2: Light-microscopic view of starch granules of wild type potatoplants.

FIG. 3: Light-microscopic view of starch granules of 072VL036 potatoplants having a reduced gene expression of the R1 and BEI gene.

FIG. 4: Light-microscopic view of starch granules of 203MH010 potatoplants according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The Following Methods were Used in the Examples

Analysis of the Starch

a) Determination of the Amylose/Amylopectin Ratio

Starch was isolated from potato plants according to standard techniquesand the ratio of amylose to amylopectin was determined using the methoddescribed by Hovenkamp-Hermelink et al. (Potato Research 31, (1988),241–246).

b) Determination of the Phosphate Content

The positions C2, C3 and C6 of the glucose units may be phosphorylatedin the starch. For determining the C6-P content of the starch, 50 mgstarch are hydrolysed in 500 μl 0.7 M HCl for 4 hours at 95° C. Then,the mixtures are centrifuged for 10 min at 15,500 g and the supernatantsare taken. 7 μl of the supernatants are mixed with 193 μl imidazolebuffer (100 mM imidazole, pH 7.4; 5 mM MgCl_(2,) 1 mM EDTA and 0.4 mMNAD). The measuring was carried out in the photometer at 340 nm. Afterestablishing a basic absorption, the enzyme reaction was started byadding 2 u glucose-6 phosphate dehydrogenase (from Leuconostocmesenteroides, Boehringer Mannheim). The change in the absorption isdirectly proportional to the concentration of the G-6-P content of thestarch.

The overall phosphate content was determined according to the method byAmes (Methods in Enzymology VIII, (1966), 115–118).

30 μl ethanolic magnesium nitrate solution are added to about 50 mgstarch and ashed for three hours at 500° C. in a muffle furnace. 300 μl0.5 M hydrochloric acid are added to the residue and incubated for 30min at 60° C. Then, an aliquot is filled to 300 μl 0.5 M hydrochloricacid, added to a mixture of 100 μl 10% ascorbic acid and 600 μl 0.42%ammonium molybdate in 2 M sulphuric acid and incubated for 20 min at 45°C.

Then, a photometric measurement is conducted at 820 nm, using aphosphate calibration series as a standard.

c) Determination of the Gel Strength (Texture Analyser)

2 g starch (TS) are dissolved in 25 ml of an aqueous 60% (w/v) CaCl₂solution and pasting is achieved in an RVA apparatus (temperatureprogram: cf. item d) “Determination of the viscosity properties by meansof a Rapid Visco Analyser (RVA)”) and then it is stored in a closedcontainer for 24 hours at room temperature. The samples are fixed undera probe (cylindrical stamp with a planar surface) of a texture analyserTA-XT2 by Stable Micro Systems (Surrey, UK) and the gel strength isdetermined using the following parameters:

test speed  0.5 mm/s depth of penetration   7 mm contact area  113 mm²pressure   2 gd) Determination of the Viscosity Properties by Means of a Rapid ViscoAnalyser (RVA)

2 g starch (TS) are added to 25 ml H₂O and used for the analysis in aRapid Visco Analyser (Newport Scientific Pty Ltd., Investment SupportGroup, Warriewod NSW 2102, Australia). The apparatus is used accordingto the manufacturer's instructions. For determining the viscosity of theaqueous solution of the starch, the starch suspension is first heated to50° C. for 1 min, then it is heated from 50° C. to 95° C. at a speed of12° C. per minute. Subsequently, the temperature is maintained at 95° C.for 2.5 min. Then, the solution is cooled down from 95° C. to 50° C. ata speed of 12° C. per minute. The viscosity is determined over the wholetime.

e) Determination of Glucose, Fructose and Sucrose

The content of glucose, fructose and sucrose is determined according tothe method described by Stitt et al. (Methods in Enzymology 174, (1989),518–552).

f) Analysis of the Distribution of the Side Chains of the Amylopectin byMeans of Ion Exchange Chromatography

For separating amylose from amylopectin, 200 mg starch are dissolved in50 ml-reaction vessels with 12 ml 90% (v/v) DMSO in H₂O. After adding 3volumes ethanol, the precipitate is separated by a 10 min-centrifugationat about 1800 g at room temperature (RT). The pellet is then washed with30 ml ethanol, dried and dissolved in 40 ml 1% (w/v) NaCl solution at75° C. After cooling down the solution to 30° C., about 90 mg thymol areadded slowly and this solution is incubated for at least 60 h at 30° C.Then, the solution is centrifuged for 30 min at 2000 g (RT). 3 volumesethanol are then added to the supernatant and the precipitatingamylopectin is separated by means of 5 min-centrifugation at 2000 g(RT). The pellet (amylopectin) is then washed with ethanol and driedusing acetone. By adding DMSO to the pellet, a 1%-solution is prepared200 μl of which are added to 345 μl water, 10 μl 0.5 M sodium acetate(pH 3.5) and 5 μl isoamylase (dilution of 1:10; Megazyme) and incubatedfor about 16 h at 37° C. An aqueous 1:5 dilution of this digestion isthen filtered with an 0.2 μm-filter and 100 μl of the filtrate areanalysed by means of ion exchange chromatography (HPAEC-PAD, Dionex).The separation is carried out with a PA-100 column (with a correspondingpre-column), the detection is carried out amperometrically. The elutionconditions are as follows:

t (min) solution A (%) solution B (%)  5 0 100 35 30 70 45 32 68 60 1000 70 100 0 72 0 100 80 0 100 stop solution A—0.15 M NaOH solution B—1 Msodium acetate in 0.15 M NaOH

The relative proportion of short side chains in the overall proportionof all side chains is determined by determining the proportion inpercent that a certain side chain has in the overall proportion of allside chains. The overall proportion of all side chains determinedthrough the determination of the overall height of the peaks whichrepresent the polymerisation degrees of DP 6 to 40 in the HPLCchromatogram. The proportion in percent that a certain side chain has inthe overall proportion of all side chains is determined by thedetermination of the ratio of the height of the peak representing thisside chain in the HPLC chromatogram to the overall height of all peakshaving a DP of 6 to 40. The program Chomeleon 6.20 by Dionex, USA wasused for determining the peak heights. The parameters of the evaluationsoftware that were to be adjusted were as follows:

retention parameter time (min) name parameter value channels 0.000Inhibit Integration on All channels 20.000 Lock Baseline on All channels20.600 Inhibit Integration off All channels 20.600 Minimum Height 0.001(Signal) All channels 45.000 Inhibit Integration on All channelsg) Determination of the Granule Size

Starch was extracted from the potato tubers according to standardmethods and washed several times with water in a 10 l-bucket (ratioheight of the bucket/diameter of the bucket=approx. 1.1). For obtainingthe starch samples which were finally subjected to the determination ofthe granule size, the starches were left to stand for about 4 h afterwashing to achieve as complete a sedimentation of the starches aspossible.

The granule size was then determined by means of a photo sedimentometerof the type “Lumosed FS1” by Retsch GmbH, Germany using the softwareV.2.3.

The software adjustments were as follows:

data of the substance: calibration no. 0 density [kg/m³] 1500sedimentation fluid: type water viscosity [Pa s] 0.001 density [kg/m³]1000 addition — measurement data 5 min sieve diameter [μm] 250 passage[%] 100 measurement range 4.34–117.39 μm calibration N temperature 20°C.

The distribution of the granule size was determined in an aqueoussolution and according to the manufacturer's instructions and based onthe literature of e.g. H. Pitsch, Korngröβenbestimmung; LABO-1988/3Fachzeitschrift für Labortechnik, Darmstadt.

h) Mechanical Fragmentation of the Strach

About 0.5 g of each starch were placed in a coffee mill (manufacturer:Mellert, type: M85, Germany) and ground six times for 30 s each. Betweentwo intervals, the grinding was interrupted for 20 s each. Thedistribution of the granule size was determined as described in item g).

i) Water Binding Capacity

For determining the water binding capacity (WBC), the supernatant wasweighed after separating the soluble portion by centrifugation of thestarch swollen at 70° C. The water binding capacity (WBC) of the starchwas set in relation to the weighed portion of the starch corrected bythe soluble mass.WBC (g/g)=(residue−(weighed portion−soluble portion))/(weighedportion−soluble portion).

The expression vector ME5/6 (cf. FIG. 1) was used in the Examples:

Preparation of the Expression Vector ME5/6

pGSV71 is a derivative of the plasmid pGSV7 which is derived from theintermediary vector pGSV1. pGSV1 is a derivative of pGSC1700 theconstruction of which has been described by Cornelissen and Vanderwiele(Nucleic Acids Research 17, (1989), 19–25). pGSV1 was obtained frompGSC1700 by deletion of the carbenicillin resistance gene as well asdeletion of the T-DNA sequences of the TL-DNA region of the plasmidpTiB6S3.

pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar etal., Gene 2, (1977), 95–113) as well as the replication origin of thePseudomonas plasmid pSV1 (Itoh et al., Plasmid 11, (1984), 206). pGSV7additionally contains the selectable marker gene aadA from thetransposon Tn1331 from Klebsiella pneumoniae which confers resistance tothe antibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24(3), (1990), 218–226; Tolmasky and Crosa, Plasmid 29 (1), (1993),31–40).

The plasmid pGSV71 was obtained by cloning a chimeric bar gene betweenthe border regions of pGSV7. The chimeric bar gene contains the promotersequence of the cauliflower mosaic virus for initiating thetranscription (Odell et al., Nature 313, (1985), 180), the bar gene fromStreptomyces hygroscopicus (Thompson et al., EMBO J. 6, (1987),2519–2523) and the 3′-non-translated region of the nopaline synthasegene of the T-DNA of pTiT37, for terminating the transcription andpolyadenylation. The bar gene confers resistance to the herbicideglufosinate ammonium.

In position 198–222, the T-DNA contains the right border sequence of theTL-DNA of the plasmid pTiB6S3 (Gielen et al., EMBO J. 3, (1984),835–846). There is a polylinker sequence between the nucleotide 223–249.The nucleotides 250–1634 contain the P35S3 promoter region of thecauliflower mosaic virus (Odell et al., cf. above). The coding sequenceof the phosphinothricine resistance gene (bar) from Streptomyceshygroscopicus (Thompson et al., 1987, cf. above) is contained betweenthe nucleotides 1635–2186. The two terminal codons at the 5′ end of thebar wild type gene were replaced by the codons ATG and GAC. There is apolylinker sequence between the nucleotides 2187–2205. The 260 bp-TaqIfragment of the non-translated 3′ end of the nopaline synthase gene(3′nos) from the T-DNA of the plasmid pTiT37 (Depicker et al., J. Mol.Appl. Genet. 1, (1982), 561–573 is located between the nucleotides 2206and 2465. The nucleotides 2466–2519 contain a polylinker sequence. Theleft border sequence of the TL-DNA from pTiB6S3 (Gielen et al., EMBO J.3, (1984), 835–846) is located between the nucleotides 2520–2544.

The vector pGSV71 was then cleaved with the enzyme PstI and blunted. Thepromoter B33 and the ocs cassette were cleaved from the vector pB33-Kanas an EcoRI-HindIII fragment, blunted and inserted into the vectorpGSV71 which had been cleaved with PstI and blunted. The vector obtainedserved as a starting vector for the construction of ME5/6. Anoligonucleotide containing the cleavage sites EcoRI, PacI, SpeI, SrI,SpeI, NotI, PacI and EcoRI was inserted into the PstI cleavage sitebetween the B33 promoter and the ocs element of the vector ME4/6 byduplicating the PstI cleavage site. The expression vector obtained wascalled ME5/6.

Description of the Vector pSK-Pac

pSK-Pac is a derivative of pSK Bluescript (Stratagene, USA) into whichPacI cleavage sites flanking the multiple cloning site (MCS) wereinserted.

The following Examples illustrate the invention:

EXAMPLE 1 Production of Transgenic Potato Plants having a Reduced GeneExpression of an R1, BEI and BEII Gene

For producing transgenic plants having a reduced activity of a BEI, R1and BEII protein, first transgenic plants were generated in which theBE1 activity and the amount of protein R1 were reduced. For thispurpose, both the T-DNA of the plasmid pB33-aR1-Hyg and the T-DNA of theplasmid pB33-a-BE1-Kan were transferred simultaneously into potatoplants using Agrobacteria as described by Rocha-Sosa et al. (EMBO J. 8,(1989), 23–29).

For constructing the plasmid pB33-aR1-Hyg and the plasmid pB33-aBE1-Kan,first the expression vectors pB33-Kan and pb33-Hyg, respectively, wereconstructed. For this purpose, the promoter of the patatin gene B33 fromSolanum tuberosum (Rocha-Sosa et al., 1989, cf. above) was ligated asDraI fragment (nucleotides −1512 to +14) into the vector pUC19 (GenBankAcc. No. M77789) which had been cleaved with SstI, the ends of saidvector having been blunted by means of the T4-DNA polymerase. In thisway, the plasmid pUC19-B33 was obtained. The B33 promoter was cleavedfrom this plasmid with EcoRI and SmaI and ligated into the vector pBinARwhich had been cleaved correspondingly. In this way, the plantexpression vector pB33-Kan was obtained. The plasmid pBinAR is aderivative of the vector plasmid pBin19 (Bevan, Nucl. Acids Research 12,(1984), 8711–8721) and was constructed by Höfgen and Willmitzer (PlantSci. 66, (1990), 221–230). Starting from plasmid pB33-Kan, theEcoRI-HindIII fragment comprising the B33 promoter, a portion of thepolylinker and the ocs terminator from pB33-Kan were cleaved and ligatedinto the vector pBIB-Hyg (Becker, Nucleic Acids Res. 18 (1), (1990),203) which had been cleaved correspondingly. As a result, pB33-Hyg wasobtained.

Then, an approximately 2000 bp-Asp718 fragment of the plasmid pRL1 whichcontains the nucleotide sequence of about +2850 to about +4850 of the R1cDNA from Solanum tuberosum (Lorberth, Charakterisierung von RL1: einneues Enzym des Stärkemetabolismus. Dissertation Freie UniversitätBerlin) in antisense orientation into the Asp718 cleavage site of theplasmid described before. The resulting plasmid was called pB33-aR1-Hyg.For constructing the plasmid pB33-aBE1-Kan, analogously to theaforementioned strategy, the promoter region of the patatin-class-I geneB33 from Solanum tuberosum—a SmaI/HindIII fragment which has a length ofabout 3100 bp and contains a partial cDNA for the BE1 enzyme from potato(Kossmann, Klonierung und funktionelle Analyse von Genen codierend füram Kohlenhydratstoffwechsel der Kartoffel beteiligte Proteine,Dissertation Technische Universität Berlin, (1992))—was first bluntedand inserted into the SmaI cleavage site of the vector pBinAR-Hyg (cf.above) in antisense orientation with regard to the B33 promoter.

After the transformation, different lines of transgenic potato plantscould be identified by means of Western blot analysis, the tubers ofsaid potatoes having a content of the R1 protein which was reducedsignificantly. Further analyses showed that isolated starch of the line36 had the highest amylose content of all transformants examinedindependently of each other.

Plants of said line were then transformed with the plasmidpGSV71-aBE2-basta as described by Rocha-Sosa et al. (EMBO J. 8 (1989),23–29).

Plasmid pGSV71-aBE2-basta was constructed by screening according tostandard procedures a tuber-specific potato cDNA library with a DNAfragment which had been amplified by RT-PCR (primer 1 (SEQ ID No. 1):5′-gggggtgttggctttgacta and primer 2 (SEQ ID No. 2)5′-cccttctcctcctaatccca; Stratagene ProSTAR™ HF Single-Tube RT-PCRsystem) with total-RNA from tubers as template. In this way, a DNAfragment which has a size of about 1250 bp (cf. SEQ ID No. 3) and whichwas then subcloned as a EcoRV-SmaI fragment into the EcoRV cleavage siteof the cloning vector pSK-Pac (cf. above) and finally ligated as PacIfragment into the expression vector ME5/6 (FIG. 1) in antisenseorientation. As a result, the plasmid pGSV71-aBE2-basta was obtained.

From plants which were obtained by the transformation with the plasmidpGSV71-aBE2-basta and which showed a reduced R1, BEI and BEII geneexpression, said plants being called 203MH plants, tissue samples oftubers of the independent transformants were taken and their amylosecontent was determined (cf. methods). The starches of the independentlines the tubers of which had the highest amylose content were used forfurther analysing the starch properties (cf. Example 2).

EXAMPLE 2 Analysis of the Starch of Plants Having a Reduced R1, BEI andBEII Gene Expression

The starch of different independent lines of the transformation 203 MHdescribed in Example 1 was isolated from potato tubers. Then, thephysico-chemical properties of this starch were analysed. The results ofthe characterisation of the modified starches are shown in Table 1(Tab. 1) for an exemplary selection of certain plant lines.

TABLE 1 phosphate RVA RVA RVA RVA RVA gel strength, in C6 amylose maxmin fin set T 60% (w/v) CaCl₂ no. genotype (%) (%) (%) (%) (%) (%) (%)(%) 1 Desiree 100 22 100 100 100 100 100 100 (wild type) 3 203MH010 2592 no pasting in H₂O approx. 17,000 5 203MH055 33 80 not measured 6203MH080 31 91 approx. 10,600 Legend: R1 = R1 enzyme, BEI = branchingenzyme I, BEII = branching enzyme II, as = antisense RVA = Rapid ViscoAnalyser, max = maximum viscosity, min = minimum viscosity fin =viscosity at the end of the measurement, set = set back = difference ofmin und fin T = pasting temperature The values in % are related to thewild type (=100%) except for the amylose content.

The distribution of the side chains of the amylopectin was analysed asdescribed above. The following Table (Tab. 2) contains an overview ofthe proportions of the individual peak heights of the HPAECchromatograms within the overall peak height of wild type plants(Desiree), of 072VL036 plants (potato plants having a reduced geneexpression of the R1 and BEI gene) and of selected lines of thetransformations 203MH (cf. Example 1: potato plants having a reducedgene expression of the R1, BEI and BEII gene):

TABLE 2 proportion of the no. of individual peak heights within theoverall peak glucose height of every potato line in % units Desi Mix072VL036 203MH10 203MH61 203MH80 dp6 2.48 1.2 2.6 1.7 2.7 dp7 1.90 1.21.6 1.1 1.5 dp8 1.46 1.4 1.0 0.8 1.2 dp9 2.48 2.2 1.3 1.1 1.5 dp10 4.383.5 2.1 1.9 2.1 dp11 6.28 4.8 3.1 2.8 3.0 dp12 7.30 5.6 3.6 3.6 3.3 dp137.88 6.1 3.6 3.9 3.3 dp14 7.88 6.3 3.9 4.4 3.6 dp15 7.30 6.3 4.2 4.4 3.3dp16 6.72 6.1 4.2 4.7 3.3 dp17 5.84 5.9 4.2 4.7 3.3 dp18 5.26 5.7 4.44.7 3.3 dp19 4.82 5.5 4.4 4.7 3.3 dp20 4.38 5.1 4.4 4.7 3.3 dp21 3.944.7 4.4 4.7 3.6 dp22 3.50 4.2 4.2 4.7 3.6 dp23 3.07 3.8 4.2 4.4 3.9 dp242.48 3.4 4.4 4.4 4.2 dp25 2.19 3.1 4.2 4.4 4.2 dp26 1.90 2.7 4.2 4.2 4.5dp27 1.75 2.4 3.9 4.2 4.5 dp28 1.31 2.0 3.6 3.6 4.5 dp29 1.02 1.6 3.43.3 4.2 dp30 0.88 1.3 2.9 2.8 3.9 dp31 0.58 1.1 2.6 2.5 3.3 dp32 0.440.8 2.1 1.9 2.7 dp33 0.29 0.7 1.8 1.7 2.4 dp34 0.29 0.5 1.8 1.4 2.4 dp350.00 0.4 1.6 1.1 1.8 dp36 0.00 0.3 1.0 0.8 1.5 dp37 0.00 0.2 0.8 0.6 0.9dp38 0.00 0.2 0.5 0.0 0.9 dp39 0.00 0.0 0.0 0.0 0.0 dp40 0.00 0.0 0.00.0 0.0 Total 100.00 100.0 100.0 100.0 100.0

If the proportion of peak heights of the individual chain lengths(indicated in DP) in the overall peak height is compared, a considerableshift towards side chains having a DP>26 can be seen with regard to thedistribution of the side chains of the amylopectin of the 203MH plantscompared to the amylopectin of wild type plants and also to 072VLplants. If the mean is calculated from the relative proportions of theside chains having a DP of 26 to DP 31, the following values areobtained (Tab. 3):

TABLE 3 Desi Mix 072VL036 203MH10 203MH61 203MH80 mean of the 1.24 1.853.43 3.43 4.15 relative proportions of DP 26 to DP 31 change in % 100149 276 276 335 compared to Desi Mix (=100%)

The amylopectin of 203MH plants is characterised by an increasedproportion of side chains having a DP of 26 to DP 31 compared toamylopectin of wild type plants and also of 072VL plants.

Furthermore, the morphology of the starch granules was examined:

The surface of the starch granules of wild type plants appears smoothunder the light microscope. The form of the granules is round to oval,no “internal structures” being noticeable. Moreover, a uniformdistribution of the different granule sizes can be seen (cf. FIG. 2).

Starch granules of 072VL036 plants (FIG. 3) have a very heterogeneousappearance. Only some granules appear smooth, others have grooves, someshow “bunch-of-grapes-like agglomerations”. Other granules havecross-recess-like structures. The spectrum of granule sizes is broad,smaller granules making up a greater proportion than is the case withstarch granules of wild type plants.

The morphology of the granules of the line 203MH010 (FIG. 4), too, isheterogeneous, though less apparent than in 072VL036 plants. The surfaceof almost all granules has grooves, most of the granules showbunch-of-grapes-like agglomerations. Sometimes, particles can be seenwhich look like fragments of these agglomerated structures. The sizedistribution is relatively broad, smaller granules dominate though.

Furthermore, the granule size was determined using a photosedimentometer of the type “Lumosed” by Retsch GmbH, Germany.

The average granule size of both untreated starch samples and sampleswhich, prior to the granule size determination, were subjected to anoverall 3-minute mechanical fragmentation was measured (for conductionsee above) (Tab. 4).

In addition, the proportion of starch granules having a size of <20 μmwas determined (Tab. 5)

TABLE 4 average granule size [μm] mechan. sample untreated fragmentationWt 23.86 22.27 072VL036 16.88 16.78 203MH010 14.78 11.31 203MH066 14.5911.82 203MH080 14.31 12.14

TABLE 5 Proportion of granules < 20 μm [%] mechan. sample untreatedfragmentation Wt 51.7 49.3 072VL036 69.7 69.9 203MH010 85.8 92.9203MH066 83.7 90.6 203MH080 88.4 91.1

The results show that both the average granule size and the proportionin percent of starch granules <20 μm of the starches of the inventiondiffer significantly from wild type starches as well as from starchesderived from 072VL036 plants.

After mechanical fragmentation of the starches, these differences areeven more significant than without mechanical treatment.

1. A transgenic plant cell which has been genetically modified, theplant cell comprising: a) in an antisense orientation relative to apromoter, a nucleic acid sequence of at least 500 nucleotides thathybridizes under high stringency conditions to a gene encoding anendogenous starch phosphorylase (R1) protein and that leads to thereduction, compared to the non-genetically modified plant cell, of theexpression of one or more endogenous starch phosphorylase (R1) proteins;b) in an antisense orientation relative to a promoter, a nucleic acidsequence of at least 500 nucleotides that hybridizes under highstringency conditions to a gene encoding an endogenous branching enzymeI (BEI) protein and that leads to the reduction, compared to thenon-genetically modified plant cell, of the expression of one or moreendogenous branching enzyme I (BEI) proteins; and c) in an antisenseorientation relative to a promoter, a nucleic acid sequence of at least500 nucleotides that hybridizes under high stringency conditions to agene encoding an endogenous branching enzyme II (BEII) protein and thatleads to the reduction, compared to the non-genetically modified plantcell, of the expression of one or more endogenous branching enzyme II(BEII) proteins.
 2. A transgenic plant cell comprising at least onenucleic acid molecule comprising: a) a nucleic acid sequence comprisingat least 500 nucleotides of a starch phosphorylase (R1) gene operablylinked to a promoter in an antisense orientation leading to reducedexpression of an endogenous gene encoding a starch phosphorylase (R1)protein compared to expression in the non-transgenic plant cell; b) anucleic acid sequence comprising at least 500 nucleotides of a branchingenzyme I (BEI) gene operably linked to a promoter in an antisenseorientation leading to reduced expression of an endogenous gene encodinga branching enzyme I (BEI) protein compared to expression in thenon-transgenic plant cell; and c) a nucleic acid sequence comprising atleast 500 nucleotides of a branching enzyme II (BEII) gene operablylinked to a promoter in an antisense orientation leading to reducedexpression of an endogenous gene encoding a branching enzyme II (BEII)protein compared to expression in the non-transgenic plant cell.
 3. Thetransgenic plant cell according to claim 1 or 2 which synthesizes amodified starch.
 4. The transgenic plant cell according to claim 3,wherein the modified starch has an amylose content of at least 75% and areduced phosphate content compared to starch from a non-transgenic plantcell.
 5. The transgenic plant cell according to claim 3, wherein themodified starch has a modified distribution of the side chains.
 6. Atransgenic plant comprising the transgenic plant cell according to claim1 or
 2. 7. A method for producing a transgenic plant cell thatsynthesizes a modified starch, comprising the step of introducing into aplant cell at least one nucleic acid molecule, wherein said at least onenucleic acid molecule comprises: a) a nucleic acid sequence comprisingat least 500 nucleotides of a starch phosphorylase (R1) gene operablylinked to a promoter in an antisense orientation leading to reducedexpression of an endogenous gene encoding a starch phosphorylase (R1)protein compared to expression in the non-transgenic plant cell; b) anucleic acid sequence comprising at least 500 nucleotides of a branchingenzyme I (BEI) gene operably linked to a promoter in an antisenseorientation leading to reduced expression of an endogenous gene encodinga branching enzyme I (BEI) protein compared to expression in thenon-transgenic plant cell; and c) a nucleic acid sequence comprising atleast 500 nucleotides of a branching enzyme II (BEII) gene operablylinked to a promoter in an antisense orientation leading to reducedexpression of an endogenous gene encoding a branching enzyme II (BEII)protein compared to expression in the non-transgenic plant cell; therebyproducing the transgenic plant cell that synthesizes a modified starch.8. The method of claim 7, wherein the modified starch has an amylosecontent of at least 75% and a reduced phosphate content in comparisonwith starch from a non-transgenic plant cell.
 9. A method for producinga transgenic plant that synthesizes a modified starch comprising thestep of regenerating a plant from a plant cell produced according to themethod of claim 7, thereby producing the transgenic plant thatsynthesizes a modified starch.
 10. The method according to claim 9,wherein the modified starch has an amylose content of at least 75% and areduced phosphate content in comparison with starch from anon-transgenic plant.
 11. A transgenic plant obtained by the methodaccording to claim 9 or
 10. 12. The transgenic plant according to claim6 which is a starch-storing plant.
 13. The transgenic plant according toclaim 12 which is a potato plant.
 14. Propagation material of the plantaccording to claim 6, wherein the propagation material comprises thetransgenic plant cell.
 15. A nucleic acid molecule comprising: a) anucleic acid sequence comprising at least 500 nucleotides of a starchphosphorylase (R1) gene operably linked to a promoter in an antisenseorientation, which when expressed in a plant cell leads to reducedexpression of an endogenous gene encoding a starch phosphorylase (R1)protein compared to a the non-transformed plant cell; b) a nucleic acidsequence comprising at least 500 nucleotides of a branching enzyme I(BEI) gene operably linked to a promoter in an antisense orientation,which when expressed in a plant cell leads to reduced expression of anendogenous gene encoding a branching enzyme I (BEI) protein compared tothe non-transformed plant cell; and c) a nucleic acid sequencecomprising at least 500 nucleotides of a branching enzyme II (BEII) geneoperably linked to a promoter in an antisense orientation, which whenexpressed in a plant cell leads to reduced expression of an endogenousgene encoding a branching enzyme II (BEII) protein compared to thenon-transformed plant cell.
 16. A host cell comprising the nucleic acidmolecule according to claim
 15. 17. The host cell according to claim 16,wherein the cell is a plant cell.
 18. The transgenic plant according toclaim 11 which is a starch-storing plant.
 19. The transgenic plantaccording to claim 18 which is a potato plant.
 20. Propagation materialof the plant according to claim 11, wherein the propagation materialcomprises the transgenic plant cell.